Research Accomplishments: Our laboratory have shown that cyclin-dependent kinase 5 Cdk5), a critical and multifunctional neuronal kinase activity is tightly regulated and involved in nervous system development, regulates large number of synaptic and other processes including survival under physiological conditions. However, due to neuronal insults is deregulated and hyperactivated in Alzheimers disease (AD) and other neuropathologies may be, in part responsible for the hallmark pathology of amyloid plaques , neurofibrillary tangles (NFTs) and neuronal death. It has been proposed that hyperactive Cdk5 results from the overexpression of p25, (a truncated fragment of p35, the normal Cdk5 regulator), which, when complexes to Cdk5, induces deregulation , hyperactivity, hyper phosphorylates tau, neurofilament and other cytoskeletal proteins forms tangles (NFTs), A-beta plaques and neuronal death. ATP analogs have been used to inhibit the kinase activity but all these compounds are non-specific and toxic. Therefore, it was important to inhibit selectively inhibit the deregulated and hyperactive Cdk5/p25 not the Cdk5/p35. Cdk5/p35 is the physiological and Cdk5/p25 the pathological targets. We asked the question what will be the effect of smaller truncated peptides of p25. This study led to the invention of a small peptide p5, a 24 amino acid selectively inhibited the Cdk5/p25 , the pathological but not Cdk5/p35 physiological activity. Recently our laboratory has shown that intraperitoneal (i.p.) injections of a modified truncated p5 peptide (TFP5), derived from the Cdk5 activator p35, penetrated the blood-brain barrier and rescued AD-like pathology in 5XFAD model mice. The principal pathology in the 5XFAD mutant, however, is extensive amyloid plaques; hence, as a proof of concept, we believe it is essential to demonstrate the peptides efficacy in a mouse model expressing high levels of p25, such as the inducible CK-p25Tg model mouse that overexpresses p25 in CamKII positive neurons. Using a modified TFP5 treatment, we show that peptide i.p. injections in these mice decrease Cdk5 hyperactivity, tau, neurofilament-M/H hyper phosphorylation, and restore synaptic function (LTP) and behavior (i.e., spatial working memory). It is noteworthy that TFP5 does not inhibit endogenous Cdk5/p35 activity, or other Cdks in vivo suggesting it might have no toxic side effects, and may serve as an excellent therapeutic candidate for neurodegenerative disorders expressing abnormally high brain levels of p25 and hyperactive Cdk5. As a proof of concept, now we have demonstrated that the peptide, injected into an AD model mouse overexpressing Cdk5/p25 (P25Tg mice), specifically targets the hyperactive kinase, reduces or eliminates AD pathology, and restores normal behavior in both in vivo and in vitro. We suggest that the peptide may serve as a potential therapeutic candidate for those neurodegenerative disorders that overexpress p25, the hyperactive activator form of p35. TP5; Protective & Restoractive Preliminary data (Sharda Yadav Current Studies) TP5 modulates the dendritic morphology Development of the neurite outgrowth and spine density is critical for the neuronal function. Previous studies revealed that Cdk5 activity is required for the development of neurite outgrowth, establishment of synaptic connections and neuronal morphology. Recently we developed , TP5 derived from the CDK5 activator p35, as an inhibitor of the hyperactive Cdk5 in vitro. To test the specificity of the TP5 we screened several kinases and noted almost 20 other kinases including Cdk5 were inhibited to some extent in test tube experiments. However, their physiological relevance has not yet established. To understand the overall impact of inhibition of these kinases by TP5 we decided to test its effect on the cultured rat cortical neurons. We focused on to observe morphological changes specifically neurite outgrowth, number of dendritic spines and establishment of the mature synaptic connections. For this cultured rat cortical neurons were transfected with ER-GFP reporter alone (to visualize the morphological changes) or in addition to Rab8-mCherry (to mark the vesicles required for neurite out growth). Neurons were treated with TP5 (500nM) from DIV3 till harvested on DIV17. Scrambled peptide (500nM) and mock treated neurons were used as negative control. Cultured neurons on the cover slips were harvested at DIV17. Neurons were fixed with 4% PFA. Images were obtained using confocal microscope LSM510. Morphological changes, number of dendritic spines were counted using Image J. We observed significant increase in the dendritic morphology of the TP5 treated cortical neurons at DIV17. We also noted a slight increase in dendritic morphology at DIV6 which was further enhance at DIV12. However, these changes were not observed in cortical neurons treated with scrambled peptide or in blank. Our results suggest that TP5 promote the neurite outgrowth in the cultured cortical neurons. TP5 enhanced the mature synaptic connections Functional synaptic connections between neurons needed for effective transfer of the electrochemical signals between neurons. Furthermore, development of functional synapses in neurons require restrained Cdk5 activity. In pathological conditions altered Cdk5 activity leads to aberrant synapse formation. To evaluate the role of the TP5 and its impact on the establishment of the synaptic connections we treated the cultured rat cortical neurons with TP5 peptide from DIV3 till DIV17. Without peptide treated neurons were used as control ( + media). Both group of the neurons were transfected with ER-GFP reporter to mark the morphological changes. We immunostained cortical neurons with synaptophysin and PSD95 (mature synaptic markers). Furthermore, quantification of the synaptophysin and PSD95 positive staining revealed an increase in the number of synaptic connection in TP5 treated neurons compared to blank (+media). Our data revealed that TP5 treated neurons establish more mature synaptic connections compared to corresponding control treated neurons. This suggest that TP5 has a role in establishing the mature synaptic connections and possibly explains part of the rescue phenotype of 5XFAD mice and p25 transgenic mice reported earlier. Future Plans; Effect of the TP5 on the transport and distribution of different organelles In neurons distribution of the different organelle is precisely regulated and represents their functional status. Aberrant organelle trafficking has been reported in several neurodegenerative disorders such as ALS, PD and AD. As Cdk5 has a wide range of substrates, many of them are involved in different cellular processes including, components of structural proteins present in different organelles, molecular motor complex protein subunits and integral part of the molecular tracks such as microtubules and their accessory factors that regulate the trafficking of the cargo from neuronal soma to axons and dendrites. Cargo trafficking occurs in both retrograde and anterograde directions and highly controlled by phosphorylation events by Cdk5. In fact, activity of the many of the anterograde and retrograde components are directly regulated by Cdk5 phosphorylation. However, the impact of the TP5 on these processes need to be further evaluated in normal and animal models of the neurodegenerative diseases. In this context, we have developed several fluorescent markers to tag different organelles (e.g. mitochondria, peroxisome, lysosome) and different vesicular compartments such as Rab5, Rab7 and Rab11). In addition, we have also obtained previously reported molecular motor complex proteins (such as Kif1a and Kif5b) fused with GFP to visualize the motors and microtubule tracks will be stained for the endogenous alpha tubulin.